1. Field of the Invention
The present invention relates to a system and method for separating particles. The invention has particular advantages in connection with separating white blood cells into desired subsets and debulking red blood cells from such white blood cells.
This application is related to U.S. Pat. No. 6,051,146 issued on Apr. 18, 2000. The entire disclosure of this U.S. patent is incorporated herein by reference to the extent it is not inconsistent.
2. Description of the Related Art
Whole blood consists of a liquid component and particle components. Sometimes, the particle components are referred to as “formed elements”. The liquid portion of blood is made up of plasma, and the particle components primarily include red blood cells (erythrocytes) (RBCs), white blood cells (WBCs), and platelets (thrombocytes). While these constituents have similar densities, their average density relationship, in order of decreasing density, is as follows: red blood cells, white blood cells, platelets, and plasma. In addition, the particle constituents are related according to size, in order of decreasing size, as follows: white blood cells, red blood cells, and platelets. The sedimentation velocities of the particle constituents are related to their size and density.
In the medical field it is often desirable to separate blood or blood components. Most current separation devices rely on density and size differences or surface chemistry characteristics to separate and/or filter blood components for transfusion or reinfusion purposes. Typically, blood components are separated or harvested from other blood components using a centrifuge. The centrifuge rotates a blood reservoir to separate components within the reservoir using centrifugal force. In use, blood enters the reservoir while it is rotating at a very rapid speed and centrifugal force stratifies the blood components, so that particular components may be separately removed. Although some centrifugal separation techniques are effective at separating some blood components from one another, many centrifugal separation processes are not capable of producing a highly purified end product.
In one type of separation procedure, white blood cells are collected by leukapheresis. Such collection typically uses a centrifuge as described above. The resulting harvested white blood cells can then be further separated into subsets of desired cells for collection if desired. Such subsets of cells desired for collection may include monocytes, lymphocytes, granulocytes, and dendritic cells, although it is understood that collection of other cells may also be desired. The collected leukapheresis products, however, are often contaminated with platelets and red blood cells which can interfere with various cell separation and/or cell selection techniques and later cultivation of the selected cells for therapeutic use.
White blood cells can also be collected by other known methods other than apheresis and again further separated into subsets of desired cells for collection.
Several methods have been proposed for the separation or fractionation of white blood cells from other particles and into selected subsets. One such method is centrifugal elutriation. In one common form of elutriation, a cell batch is introduced into a funnel-shaped chamber located in a spinning centrifuge. A flow of liquid elutriation buffer is then introduced into the chamber having the cell batch. As the flow rate of the liquid buffer solution is increased through the chamber (usually in a stepwise manner), the liquid sweeps smaller sized, slower-sedimenting cells toward an elutriation boundary within the chamber, while larger, faster-sedimenting cells migrate to an area of the chamber where the centrifugal force and the sedimentation (drag) forces are balanced.
Thus, centrifugal elutriation separates particles having different sedimentation velocities. Stoke's law describes sedimentation velocity (SV) of a spherical particle, as follows:
  SV  =            2      9        ⁢                  r        ⁢                                  ⁢        2        ⁢                  (                                    ρ              p                        -                          ρ              m                                )                ⁢        g            η      where,
r is the radius of the particle,
ρp is the density of the particle,
ρm is the density of the liquid medium,
η is the viscosity of the medium, and
g is the gravitational or centrifugal acceleration.
Because the radius of a particle is raised to the second power in the Stoke's equation and the density of the particle is not, the size of a cell, rather than its density, greatly influences its sedimentation rate. This explains why larger particles generally remain in a chamber during centrifugal elutriation, while smaller particles are released, if the particles have similar densities.
One problem with purifying white blood cells from other cells and into separate selected subsets utilizing centrifugal elutriation is that the presence of too many red blood cells in the starting white blood cell product can cause non-ideal cell separation as a result of the non-spherical shape of red blood cells and the resulting cell-cell interactions.
Another method of fractionating white blood cells from other particles and into selected subsets is the use of fluidized bed technology as disclosed in U.S. Pat. No. 5,674,173, the disclosure of which is incorporated herein by reference to the extent it is not inconsistent. Again, the presence of too many red blood cells can cause non-ideal cell separation.
To address this problem, white blood cell products in the past have initially been separated from or debulked of red blood cells by density gradient centrifugation, using various separation media. In density gradient centrifugation, a sample is layered on top of a media support and centrifuged. Under centrifugal force, the particles in the sample will sediment through the media in separate zones according to their density.
Many different types of separation media are used in density gradient centrifugation, depending upon the exact application (i.e., Sucrose, CsCl, Ficoll, Hypaque, Percol). Though available commercially, most are not FDA approved and may be deleterious to some human cell populations. The most widely used separation media is perhaps Ficoll-Paque, a solution of Ficoll and sodium diatrizoate. It is formulated to deplete the majority of granulocytes and red blood cells, while retaining a purified fraction of the mononuclear cells (lymphocytes plus monocytes). The disadvantages of using Ficoll-Paque for debulking red blood cells include the loss of 50±15% of the desired cells, and that is not currently used in a closed system.
It is know that red blood cells under proper conditions have the tendency to adhere to each other forming red blood cell rouleaux. Rouleaux formation and size, and therefore red cell sedimentation velocity, is influenced by the hematocrit of the cell suspension, exposure to shear, protein concentration, and presence of sedimentation agents.
It is against this background that the instant invention was conceived.